Worked on Protein Purification. Figure 1: Elution 1 from Purification
Figure 2 : Elution 2 from Purification
I also worked on Characterization.
Week 14:
This week I finished protein expression and finally got my Virtual screening to work. The fitness scores were pretty low, indicating that these ligands do not bind well. Run 2 is currently in progress.
Fitness Score
56.18
55.76
53.88
57.79
51.26
46.46
45.73
41.77
66.52
Figure 1: Some of the top scores from run 1
Week 13:
I began working on protein expression, using the alternate protocol of incubating for 18 hours at room temperature as opposed to 4 hours at 37 C.
I also worked on protein expression of my surrogate: FtHap. I overincubated my bacteria because I grew it overnight for 18 hours. I re-did the protocol and grew my bacteria up for exactly 12 hours, then followed that up with an 18 hour growth at room temperature. I proceeded to centrifugation and stored my pellet after adding sonication lysis buffer.
Week 11:
This week I worked on the cloning protocol. After many failed attempts at creating an accepting vector with high concentration, I used 12.6 ng/uL pNIC-Bsa4.
Figure 1: Bacterial plates. Did not spread the bacteria correctly, overincubated. No colonies resulted
Week 10
This week I worked on cloning, however the week was filled with failure. I focused on making the accepting vector for cloning, however when I did PCR clean up, my concentration for pNIC was very low each time.
Figure 1: I used 146 ng/uL pNIC, but clean up only yielded a result of 19.7.
Figure 2: Using 69.7 ng/uL pNIC (newly made to compensate for faulty 146 ng/uL), the result was 14.3 ng/uL, which is very low.
Week 9:
This week I began my cloning protocol. However, my pNIC only yielded a nanodrop result of 4.6 ng/uL.
Fig. 1: Failed pNIC
I will try this procedure again, if it fails, I will remake pNIC.
WEEK 8:
102112- Mihir, show some VS results - Dr. B This week was largely focused on doing the Virtual Screening Refresher. I did my initial runs incorrectly and spent some time making sure I ran the screenings correctly.
Week 7:
101612 - Mihir - ok good job. the PCR squared curve doesn't 'look' great - but the quanitity is high enough to justify going to cloning. Good luck. Dr. B
This week I worked on PCR squared, PCR Cleanup, and I began the Virtual Refresher.
Below is my nanodrop from my PCR squared. I had to apply wash solution, elution solution, and follow the protocol of the Sigma elution kit. After following the procedures, I was left with a small amount of eluate, which I nano dropped.
Nano Drop result
Figure 1: Nano Drop Result.
My concentration is somewhat low (31.3 ng/uL), but it is enough. My 26/280 absorbance is 1.64, and my 260/230 is .83.
Below is my result from PCR squared.
Lane 1: 100 bp ladder
Lane 2: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 1
Lane 3: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 2
Lane 4: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 3
Lane 5: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 4
Analysis of Gel: Lanes 3-5 seemed to turn out pretty well. However, lane 2 did not show up. This may be due to the fact that I used a faulty PCR machine, but that doesn't explain why all of the other lanes turned out fine. I treated all of the samples with the same conditions (temperature/mix). It could be that I may have inserted the sample into the well incorrectly (maybe I poked through the gel) resulting in the improper gel outcome.
Below is my result from secondary PCR.
Lane 1: 100 bp ladder
Lane 2: Shane
Lane 3: Akhi
Lane 4: Mihir
Analysis of Gel: The sizes seem to be fairly consistent between all three of our samples. My sample is slightly dimmer than the rest, this could be due to some error in preparation, or failure of as many primers to connect at that size.
Week 6:
100912- Mihir, ok good. Include your Secondary PCR too. - DR. B
Also, when you move on to cloning (after PCR^2) use the new T4 DNA Poly and the new dCTP and dGTP.
I worked on Primer Overlap PCR this week.
Lane 1: 100 bp ladder Lane 2: Shane's mix Lane 3: My mix Lane 4: Akhilesh's mix
The mix consisted of a reaction buffer, Hod Start Polymerase, MgSO4, 1 ul of a mix of primers (22 primers LmP, 78 ul of nanopure water), and dNTP.
Analysis of gel: My lane (lane 3) showed a spectrum of sizes, varying from small (towards the bottom) to large (top) primers. The size varies because not all of the primers join together to form the ideally long strand, and those that do are towards the top.
Week 5:
Mhir - address why you think PCR 2 didn't work. Go back and get PCR #1 to work. If you get that to work. then you can move ahead to cloning. Also, show your Primers for primer design. I don't know what to order here. -- Dr. B
This week I worked on PCR #2. Unfortunately, the gel did not show anything. Gel may not have worked due to an error in making the samples, such as insufficient, or prolonged storage.
Upstream: TACTTCCAATCCATGAAAAACTGGGTTAAAG Downstream: TATCCACCTTTACTGTTAGTACAGGTATGCTTTT Reverse Complement of the reverse primer: AAAAGCATACCTGTACTAACAGTAAAGGTGGATA
Mihir - good. may end up needing ot make more pNIC for your cloning. Good job on second attempt of PCR. You bands look good and are at the ~1,000 bp range - which is right for pGBR22. Looks like you got some carry over or contamination into the 4th lane which is negative control. Try to crop your gel images some. -- DR. B
I extracted my plasmid, pNIC-Bsa4, from my bacteria (E. Coli), using the Midiprep technique of filtration. Then I tested the concentration of my plasmid by using the Nanodrop procedure.
Figure 1: Nanodrop results of my pNIC-bsa4. My yield was 34.1 ng/uL.
PCR #1 was a failed procedure.
Figure 2: Failed PCR. DNA Ladder showed up, but none of the samples did. This could be due to errors in the mix, in the PCR, or due to the fact that the samples were not kept on ice when prepping the gel.
Redid PCR #1. Below is the result.
Figure 3. Successful gel
Lane 1: 100 bp ladder
Lane 2: Sample A (pgbr-22 template and m13 forward and reverse)
Lane 3: Sample B (same, different amount of template and water)
Lane 4: Sample C (same, different amount of template and water)
Lane 5: Sample D (same, different amount of template and water)
Analysis of Gel: The bands all seem to be the same size. The heavier bands tend to be higher up on the gel (towards the top), while the lighter bands tend to be towards the bottom. In this case, all the bands are at about the middle, indicating that their size is similar.
Week 3:
Mihir - ok good. You can move your legend for the gel to below the image (see Suman and Divyas) also include a little more analysis of gel and also include a mention of the transformation you did .-- Dr. B 091812
I worked on restriction enzyme digest. The enzymes I used were EcoR1 and Pvu-II. The buffer I used was NEBBuffer-2 (NEB stands for New England Bio Lab). Below is the result from my gel. The first lane was a skip, the second lane is the ladder, the third lane is the uncut plasmid (pgbr-22), the fourth lane is Eco-R1, the fifth lane is PVU-II, and the sixth lane is the Eco-r1+Pvu-II. Figure 1: The largeness of the top bands indicates a larger molecule. The 4th lane had one cut, the 5th lane had 2 cuts, and teh 6th lane had 3 cuts.
After RE Digestion, I worked on a practice nanodrop.
I also began my PCR #1.
Week 2:
I worked on dilution of my primer. I submitted my result to the sequencing core. Later in the week, I translated my protein onto a gel plate, using E. Coli as a control, comparing it to E. Coli with pNIC. I also worked on analyzing DNA sequence in the computer lab.
Above is the original sequence. CHOPPED: TATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANC
Above is the original sequence with many of the 'N's chopped out, because they are unreadable.
Background and Disease: S. Aureus has a role in causing food poisoning, atopic dermatitis, staphylococcal scalded skin syndrome, or Ritter's disease in neonates.
Week 15:
Worked on Protein Purification.Figure 1: Elution 1 from Purification
Figure 2 : Elution 2 from Purification
I also worked on Characterization.
Week 14:
This week I finished protein expression and finally got my Virtual screening to work. The fitness scores were pretty low, indicating that these ligands do not bind well. Run 2 is currently in progress.Week 13:
I began working on protein expression, using the alternate protocol of incubating for 18 hours at room temperature as opposed to 4 hours at 37 C.
Week 12:
112612 - Good. Dr BFigure 2: Molprobity
I also worked on protein expression of my surrogate: FtHap. I overincubated my bacteria because I grew it overnight for 18 hours. I re-did the protocol and grew my bacteria up for exactly 12 hours, then followed that up with an 18 hour growth at room temperature. I proceeded to centrifugation and stored my pellet after adding sonication lysis buffer.
Week 11:
This week I worked on the cloning protocol. After many failed attempts at creating an accepting vector with high concentration, I used 12.6 ng/uL pNIC-Bsa4.Figure 1: Bacterial plates. Did not spread the bacteria correctly, overincubated. No colonies resulted
Week 10
This week I worked on cloning, however the week was filled with failure. I focused on making the accepting vector for cloning, however when I did PCR clean up, my concentration for pNIC was very low each time.Figure 1: I used 146 ng/uL pNIC, but clean up only yielded a result of 19.7.
Figure 2: Using 69.7 ng/uL pNIC (newly made to compensate for faulty 146 ng/uL), the result was 14.3 ng/uL, which is very low.
Week 9:
This week I began my cloning protocol. However, my pNIC only yielded a nanodrop result of 4.6 ng/uL.
Fig. 1: Failed pNICI will try this procedure again, if it fails, I will remake pNIC.
WEEK 8:
102112- Mihir, show some VS results - Dr. B
This week was largely focused on doing the Virtual Screening Refresher. I did my initial runs incorrectly and spent some time making sure I ran the screenings correctly.
Week 7:
101612 - Mihir - ok good job. the PCR squared curve doesn't 'look' great - but the quanitity is high enough to justify going to cloning. Good luck. Dr. BThis week I worked on PCR squared, PCR Cleanup, and I began the Virtual Refresher.
Below is my nanodrop from my PCR squared. I had to apply wash solution, elution solution, and follow the protocol of the Sigma elution kit. After following the procedures, I was left with a small amount of eluate, which I nano dropped.
Figure 1: Nano Drop Result.
My concentration is somewhat low (31.3 ng/uL), but it is enough. My 26/280 absorbance is 1.64, and my 260/230 is .83.
Below is my result from PCR squared.
Lane 1: 100 bp ladder
Lane 2: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 1
Lane 3: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 2
Lane 4: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 3
Lane 5: 5 uL of (50 uL sample of Master Mix) + 1 uL of Blue dye- Sample 4
Analysis of Gel: Lanes 3-5 seemed to turn out pretty well. However, lane 2 did not show up. This may be due to the fact that I used a faulty PCR machine, but that doesn't explain why all of the other lanes turned out fine. I treated all of the samples with the same conditions (temperature/mix). It could be that I may have inserted the sample into the well incorrectly (maybe I poked through the gel) resulting in the improper gel outcome.
Below is my result from secondary PCR.
Lane 1: 100 bp ladder
Lane 2: Shane
Lane 3: Akhi
Lane 4: Mihir
Analysis of Gel: The sizes seem to be fairly consistent between all three of our samples. My sample is slightly dimmer than the rest, this could be due to some error in preparation, or failure of as many primers to connect at that size.
Week 6:
100912- Mihir, ok good. Include your Secondary PCR too. - DR. BAlso, when you move on to cloning (after PCR^2) use the new T4 DNA Poly and the new dCTP and dGTP.
I worked on Primer Overlap PCR this week.
Lane 1: 100 bp ladder
Lane 2: Shane's mix
Lane 3: My mix
Lane 4: Akhilesh's mix
The mix consisted of a reaction buffer, Hod Start Polymerase, MgSO4, 1 ul of a mix of primers (22 primers LmP, 78 ul of nanopure water), and dNTP.
Analysis of gel: My lane (lane 3) showed a spectrum of sizes, varying from small (towards the bottom) to large (top) primers. The size varies because not all of the primers join together to form the ideally long strand, and those that do are towards the top.
Week 5:
Mhir - address why you think PCR 2 didn't work. Go back and get PCR #1 to work. If you get that to work. then you can move ahead to cloning. Also, show your Primers for primer design. I don't know what to order here. -- Dr. BThis week I worked on PCR #2. Unfortunately, the gel did not show anything. Gel may not have worked due to an error in making the samples, such as insufficient, or prolonged storage.
Upstream: TACTTCCAATCCATGAAAAACTGGGTTAAAG
Downstream: TATCCACCTTTACTGTTAGTACAGGTATGCTTTT
Reverse Complement of the reverse primer: AAAAGCATACCTGTACTAACAGTAAAGGTGGATA
Below is my FASTA:
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTT
TAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACC
TGTACTTCCAAT CCATGAAAAACTGGGTTAAAGTTACCGGTGCGGGCGTTCTCTCTGCTACTCTGCTGCTCGGT
GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCAAACGTCAAGACCGAACAGACCCTG
AAGCCGGGTAGCCAGATCAAACTGGAAGGTGCCGTTAATGTTCGCGACCTGGGTGGTTAC
AAGACTACCGATGGTCTGACCATCAAACCGCACAAACTGATCCGCTCTGCGGAACTCGCA
AACCTCTCTGACTCTGACAAAAAGAAGCTGGTTAACACCTACGACCTGTCTCACATCGTT
GACTTCCGCACGTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTGGAC
TACACCCACGACTCTGTTATGAAAGACAACGGTACGTCTACCTCTACCCAGGACCTGACG
GCGTCTCTGGCCAAGATGGACAACCCGGAAACCTTCCTGATTAACGCGAATAAATCCTTC
ATTACCGACGAGACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTGGCGAAC
CAGGACGGTTCCGTTCTGTGGCACTGCACCGCCGGTAAAGACCGTGCCGGTTTCGGCACC
GCGCTGGTTCTGTCTGCGCTGGGTGTTGATAAAAACACCGTTATCGACGACTACATGCTG
TCTAACAAATACCGTGCTGACGAAAATAAGAAAGCGATCGAAGCGGTAGCGGCGAAAACC
GACAACAAAAAAGTGATCGACGGTATGACGGCAGTTATGGAAGTTCGTGAATCTTACATC
AACGCGGCGTTCGATGAGATCAATGCGAAATATGGTTCTATGGATAACTTCCTCAAAGAA
AAGCTGGGCCTCACTGATGCGAAGAAAGAGCAGCTGAAA AAAGCATACCTGTACTAAC
AGTAAAGGTGGATACGG
ATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCC
GGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCC
CTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATG
GGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT
GCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC
GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA
ACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG
GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATT
CTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATT
TAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGA
ACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAAC
TCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCC
GTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGC
GATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAG
AAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTT
CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTG
CGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGG
CGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATG
CTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGT
CGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTA
CCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTG
ATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGG
CCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGAC
AGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA
AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC
CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG
CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA
GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTC
TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG
CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC
GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA
CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA
GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA
CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA
ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGT
GAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC
ATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCG
CTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC
TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACC
GTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATG
TCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGC
GGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCAT
GGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGG
TTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGG
GTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCA
GATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAG
ACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCG
GTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGAT
CATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGA
CCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCG
CGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCAT
GATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGG
TTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGT
TGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC
GGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTG
ATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGA
AAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTA
CCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATC
GTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGAC
ATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGC
CAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAA
TGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTC
TGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGT
CATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACA
GGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTA
ATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACT
GTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTT
TTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCG
GCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGC
GCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTAT
GCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGG
TGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAA
GCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAA
CCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCG
AAAT
Week 4:
Mihir - good. may end up needing ot make more pNIC for your cloning. Good job on second attempt of PCR. You bands look good and are at the ~1,000 bp range - which is right for pGBR22. Looks like you got some carry over or contamination into the 4th lane which is negative control. Try to crop your gel images some. -- DR. BI extracted my plasmid, pNIC-Bsa4, from my bacteria (E. Coli), using the Midiprep technique of filtration. Then I tested the concentration of my plasmid by using the Nanodrop procedure.
Figure 1: Nanodrop results of my pNIC-bsa4. My yield was 34.1 ng/uL.
PCR #1 was a failed procedure.
Figure 2: Failed PCR. DNA Ladder showed up, but none of the samples did. This could be due to errors in the mix, in the PCR, or due to the fact that the samples were not kept on ice when prepping the gel.
Redid PCR #1. Below is the result.
Figure 3. Successful gel
Lane 1: 100 bp ladder
Lane 2: Sample A (pgbr-22 template and m13 forward and reverse)
Lane 3: Sample B (same, different amount of template and water)
Lane 4: Sample C (same, different amount of template and water)
Lane 5: Sample D (same, different amount of template and water)
Analysis of Gel: The bands all seem to be the same size. The heavier bands tend to be higher up on the gel (towards the top), while the lighter bands tend to be towards the bottom. In this case, all the bands are at about the middle, indicating that their size is similar.
Week 3:
Mihir - ok good. You can move your legend for the gel to below the image (see Suman and Divyas) also include a little more analysis of gel and also include a mention of the transformation you did .-- Dr. B 091812I worked on restriction enzyme digest. The enzymes I used were EcoR1 and Pvu-II. The buffer I used was NEBBuffer-2 (NEB stands for New England Bio Lab). Below is the result from my gel. The first lane was a skip, the second lane is the ladder, the third lane is the uncut plasmid (pgbr-22), the fourth lane is Eco-R1, the fifth lane is PVU-II, and the sixth lane is the Eco-r1+Pvu-II.
Figure 1: The largeness of the top bands indicates a larger molecule. The 4th lane had one cut, the 5th lane had 2 cuts, and teh 6th lane had 3 cuts.
After RE Digestion, I worked on a practice nanodrop.
I also began my PCR #1.
Week 2:
I worked on dilution of my primer. I submitted my result to the sequencing core. Later in the week, I translated my protein onto a gel plate, using E. Coli as a control, comparing it to E. Coli with pNIC. I also worked on analyzing DNA sequence in the computer lab.Analyzing DNA sequence:
ORIGINAL:NNNNNNNNNNNTATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANCNNNNNCNTTCNNTTNTNCNNNTNNNNNNNNNNNNNNNTTNCCGNNAGNTNNAANNGGGGNNNNNNNNNATNNGNTNNNNNNNNNNCCAAANTGNNNGNNNNGNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNGANCNNNNNNNNNNNNANNNNNNNNNNNCNNNNNAANNNNNCCNNTNNN
Above is the original sequence.
CHOPPED:
TATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANC
Above is the original sequence with many of the 'N's chopped out, because they are unreadable.
REVERSE COMPLEMENT of ORIGINAL CHOPPED:
GNTNGNNGCNGNAANTGTAGNGTCNCGCTGCGNGNNCNCNNNCCNGCCGNGCTAATGCGN
CGNTNCAGGNNGCGTCCATNGCCATCAGGCTGCGCAACTGTGGGAAGGGCGATCGGTGCG
GGCCTNTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTG
GGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATA
CGACTCACTATAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGG
GATTTTAGTGATGGTGATGGTGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCAC
ACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATCCAGTTTGCGGTCAACAT
AGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACA
AATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCAT
CTCGTGCAAAGAGACGCTCAGTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAG
GTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTAGATGAAACAGTTGCCTT
GGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCT
CCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACT
TGGTGAATGGTATGCTTCCGTATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCA
GAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCCCTCGTAAGGCTTTCCTT
TTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGT
AGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTA
GTGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTT
GAGTATNCNATA
Reverse Complement: pGEM vector
TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC
CCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAAT
AGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGG
ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG
CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA
CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTA
GTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGC
CATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG
GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTAT
AAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTA
ACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTAC
GCATCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAA
CCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAAC
CCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG
TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGC
TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGG
ATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGA
GCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGC
AACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAG
AAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGA
GTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCG
CTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGA
ATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGT
TGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACT
GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGT
TTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGG
GGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA
TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAC
TGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA
AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGT
TTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT
TTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT
GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGC
AGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTG
TAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG
ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGT
CGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAAC
TGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGG
ACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG
GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGAT
TTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTT
TACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTG
ATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAA
CGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGC
CTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGA
AAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGG
CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTC
ACACAGGAAACAGCTATGACCATGATTACGCCAAGCTATTTAGGTGACACTATAGAATAC
TCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGC
ACTAGTGATAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCC
Week 1:
We worked on target discovery and creating our target pages. Below is my target page.Organism: Staphylococcus aureus
Target: NMN phosphatase; Class B acid phosphatase precursor
EC:3.1.3.2 :Acid phosphatase
http://www.brenda-enzymes.org/literature/lit.php4?e=3.1.3.2&r=663565
Background and Disease: S. Aureus has a role in causing food poisoning, atopic dermatitis, staphylococcal scalded skin syndrome, or Ritter's disease in neonates.
Enzyme page on Brenda:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.2
Reaction pathway:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.2&Suchword=t.+cruzi&organism[]=Staphylococcus+aureus&show_tm=0
enzyme assay info: http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-acid-phosphatase.html
REAGENTS:
Sodium citrate tribasic dehydrate: 174.00
Sodium hydroxide solution: 199.00
Gene #:12865799
Essentiality of protein: None
Inhibitors: Vancomycin
Image of protein:
Coding DNA Sequence link:
http://www.ncbi.nlm.nih.gov/nuccore/JH806568.1
Amino Acid sequence:
1 mraaplllar aaslalascf cffcwldrsv lakelkfvtl vfrhgdrspi dtfptdpike
61 sswpggfggl tglgmeqhye lgeyirkryr kflndsykhe qvyirstdvd rtlmsrmtnl
121 aalfppegvs iwnpillwqp ipvhtvplse dqllylpfrn cprfqelese tlkseefqkr
181 lhpykdfiat lgklsglhgq dlfgiwskvy dplysesvhn ftlpswated tmtklrelse
241 lsllslygih kqkeksrlqg gvlvneilnh mkratqipsy kklimysahd ttvtglqmal
361 gpvipqdwst evmttnshqg tedstd
Primer Design:
1 ATGCGTGCAGCACCTCTGCTCCTGGCCCGTGCGGCCTCTCTGGCGCTGGCATCTTGC 57
2 TCTTTCGCCAGAACAGAGCGGTCCAGCCAACAGAAGAAGCAGAAGCAAGATGCCAGCGCC 60
3 GCTCTGTTCTGGCGAAAGAACTGAAGTTCGTTACTCTGGTTTTCCGTCACGGTGACCGTT 60
4 GAAGATTCTTTAATCGGATCGGTCGGAAAGGTATCGATCGGAGAACGGTCACCGTGACGG 60
5 GACCGATCCGATTAAAGAATCTTCTTGGCCGCAGGGCTTTGGTCAACTGACGCAGCTGGG 60
6 GCGCTTACGGATGTACTCGCCCAGTTCGTAGTGCTGTTCCATACCCAGCTGCGTCAGTTG 60
7 CGAGTACATCCGTAAGCGCTACCGTAAATTCCTGAACGACTCCTACAAGCATGAACAGGT 60
8 GACATGAGGGTACGGTCAACGTCAGTAGAACGAATGTAAACCTGTTCATGCTTGTAGGAG 60
9 GTTGACCGTACCCTCATGTCTCGTATGACGAACCTGGCGGCACTCTTCCCGCCTGAGGGC 60
10 ACCGGGATCGGCTGCCACAGCAGGATTGGATTCCAGATAGAGACGCCCTCAGGCGGGAAG 60
11 GCAGCCGATCCCGGTTCACACCGTTCCACTCTCTGAAGATCAACTCCTGTACCTGCCGTT 60
12 AGGGTTTCAGATTCGAGTTCCTGGAAACGCGGGCAGTTGCGGAACGGCAGGTACAGGAGT 60
13 GGAACTCGAATCTGAAACCCTGAAATCTGAGGAGTTCCAGAAACGTCTCCACCCGTACAA 60
14 CCGTGCAGGCCAGACAGTTTGCCCAGGGTCGCGATGAAGTCTTTGTACGGGTGGAGACGT 60
15 TGTCTGGCCTGCACGGTCAGGATCTGTTTGGTATCTGGTCTAAAGTTTACGACCCGCTGT 60
16 CCCAAGATGGCAGAGTGAAGTTGTGAACAGACTCGGAGTACAGCGGGTCGTAAACTTTAG 60
17 CTTCACTCTGCCATCTTGGGCGACTGAAGACACCATGACTAAACTGCGTGAGCTGTCTGA 60
18 TCTTTTTGTTTGTGGATACCGTACAGAGACAGGAGGCTCAGTTCAGACAGCTCACGCAGT 60
19 GTACGGTATCCACAAACAAAAAGAAAAATCTCGTCTGCAAGGTGGCGTTCTCGTTAATGA 60
20 GCTCGGGATCTGGGTCGCACGTTTCATGTGGTTGAGAATTTCATTAACGAGAACGCCACC 60
21 CGACCCAGATCCCGAGCTACAAAAAACTGATCATGTATTCTGCGCATGACACGACCGTTA 60
22 GCAGCAGGCCGTTGTAAACGTCCAGGGCCATCTGGAGGCCGGTAACGGTCGTGTCATGCG 60
23 TTACAACGGCCTGCTGCCGCCTTACGCCTCTTGTCACCTCACCGAACTGTACTTCGAAAA 60
24 TCTCGTTGCGATAGTACATTTCAACGAAATATTCACCTTTTTCGAAGTACAGTTCGGTGA 60
25 TGAAATGTACTATCGCAACGAGACCCAGCACGAACCGTATCCGCTGATGCTCCCAGGTTG 60
26 CCAACGAGCTCCGCGAAACGTTCCAGCGGGCAAGACGGAGAGCAACCTGGGAGCATCAGC 60
27 TCGCGGAGCTCGTTGGTCCGGTTATCCCGCAGGACTGGTCCACCGAAGTTATGACCACTA 60
28 GTCGGTAGAGTCCTCGGTGCCCTGGTGAGAGTTAGTGGTCATAACTTCGGTGG 53